Abstract: Astroviruses (AstV) are enteric viruses that are highly prevalent, causing widespread infections as evident in serological screens. Despite their medical importance, AstV are some of the least characterized enteric viruses. Indeed, until the late 2000s, it was thought that humans were only infected with one human astrovirus (HAstV) genotype comprised of eight serologically distinct serotypes (HAstV 1- 8). Pathogen discovery studies have identified at least two additional genogroups (VA and MLB), which each contains multiple strains. There is a fundamental gap in our understanding about the biology of AstV. For example, little or no information is available on the in vivo cell tropism, pathophysiology, and host responses. The long-term goal of the research program is to increase knowledge of human AstV biology and to develop effective strategies for their control and prevention. The objective of this application is to develop a robust, broadly applicable human AstV culture system for all genogroups and gain critical insights into the fundamental characteristics of AstV infection in human intestinal organoid cultures. Recent groundbreaking developments in stem cell biology have resulted in the development of novel, three-dimensional organotypic culture models. Among these are human intestinal enteroids (HIE), which are derived from stem cells isolated from human intestinal biopsy tissues or surgical resections. HIE resemble mini-intestines that contain primary human cells from multiple cell types that in culture stably maintain intestinal segment specific characteristics for long periods. Thus, this system exhibits multiple advantages over traditional transformed intestinal cell lines (e.g. Caco-2, HT29 cells). These features make HIE a novel and highly physiologically relevant model to study host - pathogen responses in the human intestine. Other enteric viruses such as human rotaviruses and human noroviruses are successfully cultured in HIE, either directly in their 3D conformation or following separation and seeding in 2D monolayers. Exciting preliminary data demonstrate replication of a VA-like AstV strain in HIE. Specifically, after infection of 2D HIE monolayers from multiple regions of the intestine, astrovirus VA1/HMO-C viral genome copies increase multiple logs over input. This increase represents viral replication, since addition of the nucleoside analogue 2'- C-methylcytidine reduced infection. To achieve our goal of adapting HIE as a robust, broadly applicable human astrovirus culture system and to begin to address the knowledge gap in human AstV biology, we will pursue the following aims: 1) Define parameters of astrovirus infection in HIE; and 2) Characterize the intrinsic and innate host responses to VA1 astrovirus infection in HIE. The approach is innovative as human AstV infection will be investigated for the first time in HIE, a physiologically relevant model of the human intestine. The impact of the research is high since it will significantly increase basic knowledge of human astrovirus-host interactions and may result in new diagnostic tools or identify novel drug targets. Studies in HIE may also identify common paradigms about how enteric viruses interact with the human intestine and how the host responds to them.